The present invention relates to viral vectors which may be used in gene delivery, such as in gene therapy, processes for viral replication, and viral vectors, cells and cell lines useful in preparation of viral vectors which may be used in gene delivery. The invention is not concerned with gene therapy itself, rather with the provision and production of vectors which may be used in gene delivery, such as in gene therapy.
The integration of therapeutic genes into specific locations of the DNA of dividing and non-dividing cells, accompanied by prolonged expression, is the optimal strategy for somatic gene therapy.
Adeno-associated virus (AAV) has the unique capacity of preferentially integrating its viral DNA within a defined region of the cellular genome, thus reducing the risks of insertional mutagenesis associated with other viruses such as retroviruses that integrate at random positions.
AAV is a non-pathogenic human parvovirus which usually requires Adenovirus (Ad) or Herpes virus as a helper to replicate efficiently. In the absence of helper virus the AAV genome integrates into host-cell genomic DNA at high frequency. Analysis of flanking sequences from latently infected cells of human origin have revealed integration of the AAV genome into a specific locus in 60-70% of cases. The integration locus (aavs1) has been sequenced and localised to human chromosome 19q13.3-qter (Kotin, R M, et al, 1990; Samulski. R J et al, 1991).
The integrated AAV genome can be rescued and replicated if cells containing an integrated provirus are superinfected with a helper virus such as Ad.
Parks et al., (1996) describe a Ad system in which a helper virus provides in trans all viral proteins required for propagation of the vector, which contains only the cis acting elements relevant for DNA replication and packaging (inverted terminal repeats and packaging signal). These two elements are contained within about 500 bp located at the end of viral genome. This allows in principle cloning into the vector of up to 37 kb of foreign DNA. Recently the complete (19 kb) human xcex11 antitrypsin genomic DNA locus has been rescued in an Ad helper dependent vector (Ad HDV) (Schiedner et al., 1998) and its administration to mice resulted in long term expression of therapeutic levels of proteins.
The AAV DNA genome is a linear single-stranded DNA molecule having a molecular weight of about 1.5xc3x97106 daltons or approximately 4680 nucleotides long. The AAV2 genome has one copy of the 145 nucleotides long inverted terminal repeat (ITR) located at each end. The AAV ITR contain palindromic sequences that can fold over to form hairpin structures that function as primers during initiation of DNA replication. Additionally, the ITRs are needed for viral integration, rescue from the host genome, and encapsidation of viral nucleic acids into mature virions. Inserted in between the ITRs of AAV there is a unique region of about 4470 nucleotides that contains two main open reading frames (ORF). The right ORF encodes three capsid proteins VP1, VP2 and VP3. These three proteins form the viral particle and are produced from transcripts controlled by promoter P40 located at map position 40. The left open reading frame of the AAV genome encodes the rep gene. Two promoters located at map positions 5 and 19 (promoters P5 and P19, respectively) control the expression of the four polypeptides derived from this ORF. Rep proteins Rep 78 and Rep 68 are produced from the P5 promoted transcripts, and Rep proteins Rep 52 and Rep 40 are synthesized from the P19 promoted transcripts. Srivastava et al. (1983) and Berns, K. I. (1996) provide the nucleotide sequence and organisation of the Adeno-Associated Virus 2 genome.
In the development of AAV vectors, it has been shown that the entire rep and cap domain can be excised and replaced with a reporter or a therapeutic transgene, and that the ITRs are the minimal signal sequence required for rescue, replication, packaging and integration of the AAV genome (Carter, B J.; Samulski J., R WO96/36364).
The development of AAV vectors for gene therapy has faced several limitations: difficulties encountered in the large scale production of replication defective recombinants, and the packaging limit of the AAV virion that cannot exceed 4.5 kb. This limitation excludes several larger genes which may be considered as potential candidates for gene therapy programmes.
Production of replication defective recombinants resulting from excision of cap and rep domain requires the cotransfection of two different complementing plasmids, one containing the gene for delivery (e.g. reporter or therapeutic gene) sandwiched between the two cis acting AAV ITRs, the second encoding the virus ORF for rep and cap peptides. Moreover, the cotransfected cell must also be infected with a helper virus (usually adenovirus or herpesvirus). While laboratory low production scale can be effectively achieved by cotransfection and infection, reproducible large scale production required for preparation of a therapeutic product is very difficult.
Additionally, recombinant AAV vectors in which rep and cap genes have been deleted do not integrate into the AAVS1 locus but they do so in a random fashion.
Targeting of integration involves the AAV rep gene products. In particular, the larger polypeptides Rep 78 and Rep 68 have been shown to bind in vitro the AAV ITRs and the aavsl, and possess helicase and site-specific endonuclease activities which may be required for AAV replication as well as AAV integration. See for example Shelling, A. N. et al (1994); Balaguxc3xa9, C., M. et al. (1997); Surosky, R. T. et al. (1997).
A possible alternative strategy is to transfer the ability for site-specific integration of AAV by transferring the appropriate viral genes and cis-acting signals required for site-specific integration into other large capacity viral vectors. Relevant elements of the AAV genome, such as the rep and cap ORF or the AAV ITRs, can be introduced into adenoviral vectors. The resulting recombinant Ad/AAV vectors would have the adeno larger packaging capacity, and the adeno characteristic ability to infect a large variety of cell types in vivo and of most eukaryotic cell lines in vitro.
A major stumbling block in the amplification of chimeric Ad/AAV vectors became apparent when it was realised that the AAV rep gene""s expression has a toxic effect on Ad (De La Maza L. M. et al., 1978; Berns, K. I., 1996). Thus, the expression of the Rep gene during virus replication in 293 cells hinders the amplification of the Ad vector, by a mechanism that is not fully understood but is associated with the disruption of the Ad replication centres, identified by immunofluorescence and in situ hybridization studies (Weitzman M. D. et al., 1996.).
To avoid this difficulty, alternative strategies have been developed where the rep peptides are provided separately and are not encoded by the Ad/AAV vector. For instance, in WO96/13598 is disclosed a hybrid Ad/AAV virus which comprises portions of an adenovirus, 5xe2x80x2 and 3xe2x80x2 ITR sequences from an AAV and a selected transgene. Additionally, the hybrid virus is linked via a polycation conjugate to an AAV rep gene (xe2x80x9chybrid virus conjugatexe2x80x9d or xe2x80x9ctransinfection particlexe2x80x9d). The major drawback of these procedures is in the low efficiency and reproducibility of the conjugation process.
Other elaborate approaches have been attempted, emphasizing that the problem with producing Ad/AAV vectors is a severe one.
The present invention is founded on the surprising realisation that a much simpler approach can be taken to produce the desired vectors in large amounts, as successfully demonstrated experimentally.
The present invention provides in various aspects new procedures for the efficient preparation of recombinant helper dependent adenoviral vectors comprising AAV rep genes, based on the finding that it is possible to avoid rep inhibition of replication of viral vectors by keeping on separate replicating units the viral replication functions required for vector amplification and the AAV rep genes.
A general method according to one aspect of the present invention includes:
(a) providing cells containing a helper adenovirus vector;
(b) introducing a helper-dependent adenoviral vector including an AAV rep gene into the cells.
AAV rep 78 and/or rep 68 may be included in the helper-dependent adenoviral vector (Surosky, R. T. et al. 1997). rep50 and/or rep42 may additionaly be included. Modified forms of a rep gene which may encode a modified protein may be used, provided the ability of the protein to promote nucleic acid integration into the chromosome is retained. A modified form which is inactive or substantially inactive unless activated by means of an appropriate stimulus or signal may be employed.
Generally such a method is employed in production of adenoviral particles containing nucleic acid including the AAV rep gene, so that the method further includes incubating or cultivating the cells under conditions and for an appropriate length of time for production of such adenoviral particles. Adenoviral particles that are produced may be harvested, may be isolated and/or purified, and may be used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a composition (pharmaceutical) which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e.g. see below).
A substantially improved and preferred method further includes an additional step of pre-incubating the helper virus vector containing cells, for a period of time sufficient to allow expression of viral proteins required for Ad genome replication, before introducing the helper-dependent adenovirus vectors including an AAV rep gene.
Methods in accordance with the present invention are generally applicable to the preparation of vectors that by carrying one or more AAV rep genes have the property to inhibit adenovirus replication.
In a preferred method the helper is a helper adenovirus, and may be AdLC8cluc (Parks et al., 1996).
The optimal concentration of helper to be used in the method may vary, and if the helper virus genome is introduced into the host cell by DNA transfer methods, for each method a range of concentration may be used. If the helper genome is delivered by the use of infective viral particles, generally the range of concentration may be between 1 and 100 m.o.i. (multiplicity of infection), the preferred concentration being between 1 and 5 m.o.i.
The rep gene is or rep genes are generally under the control of a promoter, and most preferably under the control of a regulatable promoter. Suitable promoters include the xcex11-anti trypsin promoter, T7 promoter, and tissue-specific promoters. T7 promoter has a very low activity in the absence of the phage T7 polymerase and is an appropriate promoter to rescue adenovectors encoding toxic genes.
In preferred embodiments, the promoter is a tissue specific promoter that is known to be active in the cells of the target tissue (e.g. liver) but less active in the cell chosen as packaging cells for the production of the viral adenovectors.
The preparation of helper competent cells may achieved by infection of suitable cell lines with infective viral particles containing the helper virus genome. Alternatively, the helper virus genome may be delivered into the host cell using a variety of DNA transfer methods such as electroporation, DEAE-dextran, calcium phosphate, DNA gun, liposomes, etc.
The cell line that may be used as host is any cell line capable to sustain the replication of the helper and of the vector to be amplified. A preferred cell line is 293 for its ability to sustain defective adenovirus replication, in particular the 293 derived line 293cre (Chen L., et al., 1996). Use of this cell line selectively favors the packaging and amplification of helper dependent vectors which do not contain loxP sites at either end of the Adenovirus packaging signal. In contrast, packaging of a helper adenovirus such as AdLc8cluc is inhibited in this cell line, where the helper virus has been engineered to carry two loxP sites substrates of the Cre recombinase flanking the packaging signal (Parks et al. (1996) supra).
Instead of the cre-loxP recombinase system, other helper vectors may be employed based on the use of different recombinases and their specific substrate, and of suitable host cell lines. For example the yeast FLP recombinase and its recombinase sites may be used (O""Gorman et al. 1991).
In a preferred embodiment the invention relates to a selective timing of the delivery of the helper and of the helper-dependent vector to the same host cell to avoid replication inhibition due to AAV rep expression. An important part of this approach is the incubation time which extends from the time the helper virus is introduced into the cell line until the time the helper dependent adenoviral vector containing the rep gene is introduced into the cell line.
Generally this time should be sufficient for adenovirus early genes expression under the cultivation conditions. Standard conditions may be used, such as wherein infected 293cre cells are maintained in minimal essential medium (MEM) supplemented with 5% Horse serum (HS), 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. Cells are grown in tissue culture dishes (Falcon) at 37xc2x0 C. in 5% CO2.
Cells may be cultured for approximately between 0.5 and 12 hours, more preferably at least about 2 hours, maybe about 2-6 hours, or about 3-7 hours, and more preferably about 4 hours. Optimal timing may be established by monitoring the final titre in defective vector obtained, for example by setting up a series of growth experiment in parallel. For the preparation of viral particles containing a defective viral vector comprising an AAV rep gene, the viral vector genome is usually delivered to the helper infected cells by transfection in a first cycle, and by infection with infective viral particles containing the defective virus genome in successive cycles of amplification of the viral vector. (See e.g. Parks et al. 1996 supra.) Other DNA transfer method may be used, such as electroporation, DEAE-dextran, calcium phosphate, DNA gun, liposomes, etc.
Generally in the present invention, the viral vector is a recombinant Ad/AAV vector including one or more of the AAV rep genes, alone or in combination, under the control of a single or multiple regulatory elements. A preferred vector may be one selected from the group consisting of pRS1032; pRS1033; pRA1034 and pSTK (Schiedner et al., 1998) (see experimental section below).
The present invention also provides in further aspects host cells and cell lines infected with an Ad/AAV vector and Ad helper virus in accordance with the present invention.
The present invention also provides a system for replication and packaging in cultured cells of recombinant DNA into mature virions. Viral stocks containing recombinant DNA encapsidated into mature virions obtained by the methods here described represent further aspects of the present invention and can be used to transfer genetic information into any cell or tissue of choice.
Nucleic acid sequences can be readily prepared and manipulated by the skilled person using the information and references contained herein and techniques known in the art. In order to obtain expression of desired nucleic acid sequences for delivery, the sequences can be incorporated in a vector in accordance with the present invention having control sequences operably linked to the nucleic acid to control its expression. The vectors may include sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, and may include one or more further sequences so that the polypeptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell. Suitable regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate, are available in the art. For further details see, for example, Sambrook et al., 1989. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al. eds., 1992. Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying cells containing nucleic acid of interest, as is well known in the art.
Adenoviral particles containing nucleic acid encoding an AAV rep gene produced in accordance with the present invention may be formulated in pharmaceutical compositions. These compositions may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer""s Injection, Lactated Ringer""s Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Following production of adenoviral particles containing nucleic acid encoding an AAV rep gene in according with the present invention, and optional formulation of such particles into compositions, the particles may be administered to an individual, particularly human or other primate.
This may be for a therapeutic purpose, e.g. in delivery of a functional gene encoding an authentic biologically active product in a method of gene therapy, to treat a patient who is unable to synthesize that product or unable to synthesize it at the normal level or in normal form, thereby providing the effect provided by the wild-type and ameliorating one or more symptoms of the relevant disease. Examples include provision of Factor VIII to a haemophiliac, or erythropoietin to an individual with chronic anaemia, chronic renal failure or sickle cell anaemia. Other therapeutically useful genes include those encoded Factor IX coagulation factor, LDL-receptor, insulin, dystrophin and CFTR.
Administration is preferably in a xe2x80x9cprophylactically effective amountxe2x80x9d or a xe2x80x9ctherapeutically effective amountxe2x80x9d (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington""s Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.
A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
Aspects and embodiments of the present invention will now be illustrated further with reference to exeperimental exemplification and the following figures. Further aspects and embodiments will be apparent to those of ordinary skill in the art.
The term xe2x80x9ccomprisexe2x80x9d is used herein in the sense of xe2x80x9cincludexe2x80x9d.